Isolation and initial characterization of the BRCA2 promoter.

The hereditary breast cancer susceptibility gene, BRCA2, is considered to be a tumor suppressor gene that may be involved in the cellular response to DNA damage. The transcript for this gene is cell cycle regulated with mRNA levels reaching a peak just before the onset of DNA synthesis. In order to define the mechanisms by which BRCA2 is transcriptionally regulated, we have begun to study upstream regulatory sequences. In this report, we define a minimal promoter region that has strong activity in human breast epithelial cells. Deletions of this sequence narrowed the strong basal activity to a region extending from -66 to +129 with respect to the BRCA2 transcriptional start site. This sequence demonstrated cell cycle regulated activity with kinetics similar to the endogenous transcript. Examination of the sequence revealed several consensus binding sites for transcription factors including an E-box, E2F and Ets recognition motifs. Electrohoretic mobility shift assays revealed specific protein binding to two sequences upstream of the start site; the palindromic E-box and an Ets/E2F site. Site-directed mutagenesis of either of these sites reduced both the basal activity in log phase cells and the cell cycle regulated activity of the promoter. Mutational inactivation of both sites within the same construct effectively eliminated promoter activity. Antibodies to candidate transcription factors used in super shift experiments revealed specific interactions between the BRCA2 promoter and the basic region/helix - loop - helix containing USF-1 and 2 proteins and Elf-1, an Ets domain protein. Binding of these factors depended upon the presence of intact recognition sequences. The USF factors were shown to bind predominantly as a heterodimeric complex of USF-1 and 2 while Elf-1 bound the promoter when it was not occupied by USF. Co-transfection studies with USF proteins and the varicella zoster IE62 protein provide evidence for the involvement of endogenous and exogenous USF in the activation of the BRCA2 promoter. We propose that interactions between USF-1, USF-2 and Elf-1 play an important role in the transcriptional regulation of the BRCA2 gene.

BRCA1 expression is not directly responsive to estrogen.

Expression of the breast cancer susceptibility gene, BRCA1, is induced by 17-beta estradiol (E2) in estrogen receptor containing breast cancer cell lines. Our previous studies have shown that BRCA1 transcription is also regulated with the cell cycle, reaching maximal levels just before the onset of DNA synthesis. In this study, we have examined whether the estrogen induction of BRCA1 is direct or is a result of the mitogenic activity of the hormone. Four lines of evidence lead us to conclude that E2 induces BRCA1 primarily through an increase in DNA synthesis: (1) The kinetics and magnitude of induction are different from the directly E2 inducible gene, pS2; (2) Induction of BRCA1, but not pS2, is blocked by cycloheximide indicating that de novo protein synthesis is required; (3) Other hormonal and growth factor treatments that induce DNA synthesis have a similar effect, including IGF-1, EGF and DNA synthetic flares induced by tamoxifen and retinoic acid; (4) BRCA1 genomic fragments near the 5' end of the gene containing putative estrogen response elements fail to respond to E2 when transfected into breast cancer cell lines. The most consistent explanation for these findings and other published studies is that BRCA1 transcription is induced as a result of the mitogenic activity of E2 in estrogen receptor positive cells.

Relationship between p21 expression and mutation of the p53 tumor suppressor gene in normal and malignant ovarian epithelial cells.

In many cell types, p53-mediated growth inhibition is dependent on induction of p21, which is an inhibitor of cyclin-dependent kinases that are required for cell cycle progression. Failure of mutant p53 proteins to transactivate p21 may lead to uncontrolled proliferation. Because many ovarian cancers have mutations in the p53 gene, we examined p21 levels in normal and malignant ovarian epithelial cells to determine whether p21 expression is dependent on wild-type p53. Normal ovarian epithelial cells and two ovarian cancer cell lines with wild-type p53 expressed readily detectable levels of p21, whereas in p53 null and mutant cell lines, expression of p21 was diminished strikingly. A correlation between the status of the p53 gene and p21 expression also was noted in 23 primary epithelial ovarian cancers. Normal levels of p21 RNA were seen in 4/7 (57%) cancers with wild-type p53, whereas 14/16 (88%) cancers with mutant p53 had reduced p21 expression (P < 0.05). In addition, we found that lambda-irradiation of normal and malignant ovarian epithelial cells with wild-type, but not mutant, p53 resulted in induction of p21. These data are suggestive that induction of p21 is a feature of p53-mediated growth inhibition in normal ovarian epithelial cells. Conversely, mutation of the p53 gene in ovarian cancers usually is associated with decreased p21 expression. The lack of an absolute correlation between p21 expression and the status of the p53 gene in ovarian cancers is consistent with other studies that have suggested that p21 may also be regulated by p53-independent pathways.

Causes of windowing-induced dysmorphogenesis (neural tube defects and early amnion deficit spectrum) in chicken embryos.

Previous reports suggest that windowing the shells of chicken eggs during the first day of incubation frequently results in dysmorphogenesis of the central nervous system. We report here data that further delineate the neural tube defects associated with this animal model. In addition, we describe another birth defect syndrome associated with windowing: the early amnion deficit spectrum (EADS). Several components of the egg are altered structurally by windowing: the shell, outer and inner shell membranes, yolk, and air space at the blunt end of the egg. A new air space is formed over the embryo as the original one at the blunt end is obliterated. A series of studies (pH, oxygen and carbon dioxide tensions, relative humidity, temperature, and deformation of the yolk documented with magnetic resonance imaging) examining individual steps of the windowing procedure and additional techniques that stimulate windowing suggest that mechanical stress causes isolated neural tube defects and dehydration causes amnion defects. These amnion defects are associated with other embryonic abnormalities suggestive of deformations consistent with EADS.

The effects of orientation on volume measurements of human left ventricular casts.

Biplane angiocardiography has emerged as a standard method for calculating left ventricular volume measurements. Validation of such measurements has separately been made for several projections of silicone casts of the left ventricle. This study was undertaken to establish and compare the regression lines for left ventricular cast volume measurements in the commonly used angiographic projections. Once established, any ventricular volume can be calculated from the slope and intercept of the regression line obtained from the appropriate angiographic projection.

1981 Memorial Award Paper. Detectability of hepatomas in rat livers by nuclear magnetic resonance imaging.

NMR imaging of rats with implanted hepatomas in the liver demonstrates that under the imaging conditions of this study tumors of over 8 mm2 in area can be detected with high accuracy. Compared to normal liver, approximately 70% of these tumors had a combination of relaxation times (T1-T2) that could be uniquely identified as tumors, while the other 30% demonstrated relaxation time combinations that overlapped those previously found for abscesses, brain, and hematomas.

Indicator dilution time-activity curves demonstrated by rapid magnetic resonance imaging techniques and paramagnetic contrast agent.

Indicator dilution time-activity curves are demonstrated using magnetic resonance imaging (MRI) as the detector and a paramagnetic contrast agent as the indicator. Manganese chloride was injected into a flow phantom. The nuclear magnetic resonance (MR) intensity was measured downstream. Several flow rates were used. The observed MR intensity decreased as the paramagnetic indicator passed through the imaging plane. The qualitative changes of the MR intensity decrease varied in accordance with indicator dilution theory. The equations for gradient refocused echoes, paramagnetic compound relaxation changes, and the indicator dilution analysis were combined and evaluated. Quantitative analysis demonstrates several problems in its implementation.

Interactions of paramagnetic contrast agents and the spin echo pulse sequence.

The theoretical equations for paramagnetic contrast agent effects and the spin echo pulse sequence are combined to graph magnetic resonance (MR) intensity as a function of paramagnetic contrast agent concentration for various tissues. Analysis of the graphs and equations demonstrate several technical and clinical implications. These include: (1) positive enhancement is most likely to occur with short TEs and TRs; (2) changes in machine parameters TE and TR will change the concentration of agent at which the peak enhancing MR intensity will occur; (3) there is an absolute maximum MR intensity that can be reached with contrast enhancement; (4) the maximum MR intensity reached with enhancement is dependent on the tissues' T2 and, to a lesser degree, T1 relaxation times; (5) certain TE and TR combinations will cause no enhancement; (6) if positive enhancement does occur, it will usually occur only over a limited range of agent concentration; and (7) the tissues' T1 relaxation time but not its T2 time determines whether positive enhancement will occur and the relative amount of enhancement.

Use of nitroxides as NMR contrast enhancing agents for joints.

NMR imaging is a well-established technology for obtaining cross-sectional anatomic pictures of organs and tissues. In addition, NMR can provide valuable information about the physiologic state of organs and tissues, especially, as a consequence of cellular injury. With this in mind, NMR in combination with gadolinium-based contrast enhancing agents has been used to assist in the detection of abnormalities to joints as well as to evaluate the status of damage resulting from an injury to this site. We describe the synthesis of a new nitroxide, which is bioresistant to the one-electron reduction mediated by superoxide in the presence of cysteine. This model mimics the reduction of nitroxides by extracellular secretion of superoxide by PMA-stimulated neutrophils. With this nitroxide, we found, in the range from 15 to 17.5 mumoles, enhancement of an NMR image in the knee joint of rabbits. Of interest is the finding that the contrast image remained for at least 90 minutes. These results demonstrate the utility of nitroxides as contrast enhancing agents for NMR imaging of joints.

Magnetic resonance imaging of the adrenal glands.

In this article, the authors provide a discussion of how magnetic resonance imaging (MRI) is used to image the adrenal glands, how the glands appear on MR images, and what pathology can be detected at present. Although experience with MRI of the adrenal glands is still limited, the authors anticipate a promising future for the technique in this area because of its lack of ionizing radiation and its ability to easily demonstrate blood vessels.

Comparison of function and fixation of small incision circular and oval poly(methyl methacrylate) intraocular lenses.

A prospective study of function and fixation was done on 40 patients implanted with small incision poly(methyl methacrylate), single piece, biconvex intraocular lenses after in situ phacoemulsification cataract extraction via a continuous curvilinear capsulorhexis. A 5.0 mm circular optic (Pharmacia 740 P), 14 mm length lens was compared with an oval 5.0 x 6.0 mm optic (IntraOptics SP18UB), 12.75 mm lens. Twenty patients were implanted with each design and were surveyed three months postsurgery. Satisfactory visual acuity and absence of motor vehicle headlight glare was found in all 40 patients. One glare disability of 35% occurred in a patient with an off-center capsulorhexis that formed a Soemmering's ring partially posterior to a circular optic lens. Fixation was clinically central in all patients. A space between optic and posterior capsule persisted in seven eyes with an oval optic lens and in eight eyes with a circular optic lens. Therefore both designs were associated with a "no space, no cells" situation that should decrease eventual pearl formation. Total Soemmering's rings formed posterior to 9 of 20 circular optic lenses if the capsulorhexis was 5 mm or larger in diameter (only one oval optic lens formed a Soemmering's ring). Compression of the posterior capsule occurred in seven of the nine patients with circular optic lenses and total Soemmering's rings. I conclude that the circular optic design was best implanted via a 5 mm or slightly larger central capsulorhexis to promote both Soemmering's ring formation and posterior capsule compression. Both designs provided satisfactory clinical results.

Anterior chamber metal fragments after phacoemulsification surgery.

PURPOSE: To determine whether metal fragments can be shaken loose from phaco needles during surgery and embed in the iris. SETTING: Private practice, Vernon, British Columbia, Canada, and scanning electron microscope laboratory, Mastel Precision, Rapid City, South Dakota, USA. METHODS: The surfaces and rims of new and used phaco needles and the lumens of halved new needles were examined by scanning electron microscopy (SEM). To determine whether the fragments on the phaco needles were approximately the same size as those seen in the iris, a photograph of an eye with metal fragments imbedded in the iris was projected and the image size of the metal fragments approximated by using their magnification value. The magnification scale of the SEM images was used to determine the size of the metal fragments photographed on the phaco needles. RESULTS: The SEM studies of new phaco needles revealed tiny fragments of metal firmly adherent to the interior, exterior, and rim surfaces. No fragments were detected on the surfaces of the used phaco needles. Two metal fragments in the eye photograph were calculated to be 0.20 x 0.20 mm and 0.15 x 0.20 mm. Those in the SEM photos were calculated to be 0.03 to 0.10 mm. CONCLUSION: Althogh SEM of new titanium phaco needles revealed adherent metal fragments on their lathed surfaces, no fragments were found on used phaco needles. The iris fragments calculated from a projected photograph were slightly larger than those from the SEM micrographs, supporting the conclusion that annealed metal fragments shook loose from the phaco needles. This indicates that ultrasonic activation of a new phaco needle with metal fragments annealed to its surface causes fragments to release and embed in the iris.

Complex response of breast epithelial cell lines to topoisomerase inhibitors.

The topoisomerase inhibitors, camptothecin and etoposide target the activity of topoisomerase I and II respectively. These agents, or their analogues, are undergoing clinical trials for the treatment of metastatic breast cancer. In this study, we examined the response of eight breast epithelial cell lines, including six lines derived from breast cancers and two immortalized normal epithelial lines to camptothecin and etoposide. The lines varied by 700 fold in their sensitivity to the growth inhibiting effects of camptothecin and 30 fold in their response to etoposide. The BT474 line was the most resistant to both agents. The other cell lines did not have uniform sensitivity to both drugs, i.e., some lines were sensitive to one drug but relatively resistant to the other. A variety of parameters in these lines were analyzed to elucidate mechanisms of resistance including S phase, doubling time, expression and activity of topoisomerase I and II, expression of mdr-1, p53 status, cell cycle arrest, level of apoptosis, and expression of the apoptotic proteins Bcl-2 and Bax. We found that low levels of the topo I protein and its enzymatic activity were associated with increased resistance to camptothecin. This was not true for topo II activity and etoposide. Increased apoptotic responses were generally observed in cell lines that were sensitive to etoposide and this correlated with low ratios of Bcl-2/Bax protein. No single parameter was entirely predictive of response. However, the BT474 line displayed a series of characteristics including slow growth, the presence of mutant p53, low topo I activity, and a high Bcl-2/Bax ratio which together likely contributed to the resistance of this line to both etoposide and camptothecin.

Magnetic resonance imaging in breast cancer staging.

For many solid carcinomas, high-resolution cross-sectional imaging has changed cancer staging, the evaluation of therapeutic response, the detection of recurrence, and even how therapy is selected and performed. Such imaging has not yet had similar effects on breast cancer. Evaluations of therapeutic response in breast carcinomas have been impeded by the current limited methods of evaluating breast tumor size and extent: clinical palpation, ultrasonography, and mammography. The use of magnetic resonance imaging (MRI) of the breast in the evaluation of breast tumors brings the advantages of high-resolution cross-sectional imaging to breast cancer staging and treatment evaluation and is likely to greatly enhance research efforts in this complex disease. MRI of the breast has evolved to be the most accurate noninvasive technique for local staging of breast cancer. MRI is most accurate in measuring tumor size and detecting multicentric disease. These staging characteristics affect the selection of therapy and initial determination of prognosis; therefore, MRI of the breast can change the assessment of fundamental parameters on which treatment is selected. Because clinical trials of new cancer treatments are predicated on proper and accurate characterization of the tumor, MRI also should affect how clinical trials are performed and evaluated.

MR imaging of multiple hepatic cysts in a patient with polycystic liver disease.

We report a case of a 55-year-old woman with polycystic liver disease that we examined with magnetic resonance (MR) imaging, CT and ultrasound. The MR images demonstrated varying intensities in the cyst contents which we believe is due to different degrees and age of hemorrhage. Such variability among the cysts was not seen on the CT or ultrasound images.

Magnetic resonance imaging can cause focal heating in a nonuniform phantom.

To test if the radiofrequency fields of a magnetic resonance imager could cause focal heating, two cylindrical phantoms were made from a mixture of agar and saline. The first phantom was uniform; the second was nonuniform in that a narrow bridge of agar was produced. Both phantoms were exposed to high levels of radiofrequency power (140 W) at 63 MHz and the temperature rises were measured. In the uniform phantom, the temperature increased as the radius increased. In the bridge phantom, the narrow bridge heated three times greater than at the opposite uniform periphery, and over five times the average of the uniform phantom. This experiment demonstrates that the radiofrequency fields of magnetic resonance imagers can cause focal heating if the exposed object is nonuniform. Since nonuniformity is present in the human body, as the radiofrequency power of magnetic resonance imaging techniques increases, focal heating in patients is a concern.

Assessing the potential versus the actual earnings of academic radiologists: effects of unequal duty service assignments.

RATIONALE AND OBJECTIVES: The purpose of this study was to determine if annual total work relative value units (RVUs) can be used to accurately compare physician productivity and effort among a small group of similarly trained radiologists. MATERIALS AND METHODS: The annual procedures for nine abdominal imaging radiologists were obtained. The work RVU was assigned to each procedure and summed for each radiologist. The daily work RVU mean earnings by duty service (eg, ultrasound [US], gastrointestinal radiology) were calculated for each radiologist and for the entire group. RESULTS: Annual total work RVUs earned by the six full-time radiologists ranged widely (5,000 to >9,000). Mean work RVUs earned per day by all the radiologists for each duty service also ranged widely (74 for US vs 23 for gastrointestinal radiology). The range of mean work RVUs earned per day by the radiologists within each duty service was narrower, however, and had almost no statistical significance. The wider range of annual total work RVUs earned by the radiologists resulted primarily from unequal distribution of duty service assignments. For example, radiologists with more days spent performing gastrointestinal radiology had lower annual total RVUs compared to radiologists with more days spent performing computed tomography or US. CONCLUSION: The RVU is an accurate measure of income production but may be an inaccurate measure of effort and individual productivity because of differences in duty assignments. In a relatively homogeneous group of radiologists/practitioners, such a comparison should be done within a duty service, or a correcting methodology should be used, because assignment to duty services rarely is equalized across physicians.

Technologic considerations for breast tumor size assessment.

Breast tumor staging and treatment response is primarily determined by tumor size. Presently there are four techniques to measure breast cancer size: (1) palpations, (2) mammography, (3) ultrasonography, and (4) MR imaging. This article reviews the applications and limitations of each technique and the improvements MR imaging can provide.

Herpes viral hepatitis in a toucan.

Herpesvirus infection was diagnosed in a toucan. The herpesvirus was isolated from the liver and identified by electron microscopy in the liver and in cell culture. A negative immunofluorescent reaction was obtained when virus-infected cell cultures were reacted with a conjugate to the herpesvirus of Pacheco's disease. The main pathologic finding in the toucan consisted of a severe necrotizing hepatitis with intranuclear inclusions in the liver and spleen. A presumptive diagnosis of chlamydiosis was also made, based on a positive direct fluorescent monoclonal antibody reaction to chlamydial antigens in impression smears of liver and spleen. Chlamydial isolation attempts were unsuccessful. The toucan had been in contact with two macaws that had died 5 days before the toucan died and were diagnosed by histology as having herpesvirus hepatitis.

Endogenous isopropanol: forensic and biochemical implications.

Concomitant findings of isopropanol and acetone in biospecimens of decedents known not to have been exposed to the alcohol prompted a study to explain its origins. Mixtures of acetone, ADH, and NADH at pHs 7.3 and 8.8 were incubated at 37 degrees C for varying intervals. Reaction products were then analyzed by headspace GC and assured identification made by GC/MS. It was found that isopropanol is produced by reduction of acetone at pH 7.3 (to a lesser extent at pH 8.8), providing evidence for an alternate metabolic route for acetone. A mechanism for this reduction is proposed. Ranges for isopropanol (in mg/dL or mg/100 g) found in unexposed decedents were: blood 1-29; liver 7-59; brain 2-12; kidney 6-26. Thus, the forensic investigator must interpret isopropanol results cautiously, particularly when low concentrations are found.